期刊
ACS PHOTONICS
卷 7, 期 4, 页码 1036-1049出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsphotonics.9b01749
关键词
nanocrystal; nanoparticle; nonlinear microscopy; second-harmonic generation; single-plane illumination microscopy; zebrafish
类别
资金
- Agence Nationale de la Recherche [ANR-11-EQPX-0029 Morphoscope2, ANR-2010-JCJC-1510-01, ANR-10-INBS-04]
- (France BioImaging)
- European Union [268379]
- Ecole Polytechnique
- Region Ile-de-France
- Royal Society Wolfson Research Merit Award
Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structures occurs predominantly along the illumination beam. By contrast, point-like sources such as SHG nanocrystals can efficiently scatter light in multiple directions and be detected using the orthogonal geometry of a light-sheet microscope. This study investigates the suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging of SHG nanoprobes. Parameters that govern the detection efficiency of KTiOPO4 and BaTiO3 nanocrystals using SHG-SPIM are investigated theoretically and experimentally. The effects of incident polarization, detection numerical aperture, nanocrystal rotational motion, and second-order susceptibility tensor symmetries on the detectability of SHG nanoprobes in this specific geometry are clarified. Guidelines for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon excited fluorescence. Finally, microangiography was achieved in live zebrafish embryos by SHG imaging at up to 180 frames per second and single-particle tracking of SHG nanoprobes in the blood flow.
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